RNA Sequencing Quantitative Analysis and identification of RNA editing sites of Wild Type and ADAR1 editing deficient (ADAR1E861A) murine fetal liver RNA

Purpose: RNA editing by ADAR1 is essential for hematopoietic development. The goals of this study were firstly, to identify ADAR1-specific RNA-editing sites by indentifying A-to-I (G) mismatches in RNA-seq data compared to mm9 reference genome in wild type mice that were not edited or reduced in editing frequency in ADAR1E861A editing deficient mice. Secondly, to determine the transcriptional consequence of an absence of ADAR1-mediated A-to-I editing. Methods: Fetal liver mRNA profiles of embryonic day 12.5 wild-type (WT) and ADAR1 editing-deficient (ADAR1E861A) mice were generated by RNA sequencing, in triplicate (biological replicates), using Illumina HiSeq2000. The sequence reads that passed quality filters were analyzed at the transcript level with TopHat followed by Cufflinks. qRT–PCR validation was performed using SYBR Green assays. A-to-I (G) RNA editing sites were identified as previously described by Ramaswami G. et al., Nature Methods, 2012 using Burrows–Wheeler Aligner (BWA) followed by ANOVA (ANOVA). RNA editing sites were confirmed by Sanger sequencing. Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome (build mm9) and identified 14,484 transcripts in the fetal livers of WT and ADAR1E861A mice with BWA. RNA-seq data had a goodness of fit (R2) of >0.94 between biological triplicates per genotype. Approximately 4.4% of the transcripts showed differential expression between the WT and ADAR1E861A fetal liver, with a LogFC≥1.5 and p value <0.05. A profound upregulation of interferon stimulated genes were found to be massively upregulated (up to 11 logFC) in ADAR1E861A fetal liver compared to WT. 6,012 A-to-I RNA editing sites were identified when assessing mismatches in RNA-seq data of WT and ADAR1E861A fetal liver. Conclusions: Our study represents the first detailed analysis of fetal liver transcriptomes and A-to-I RNA editing sites, with biologic replicates, generated by RNA-seq technology. A-to-I RNA editing is the essential function of ADAR1 and is required to suppress interferon signaling to endogenous RNA. Fetal liver mRNA profiles of E12.5 wild type (WT) and ADAR E861A mutant mice were generated by deep sequencing, in triplicate, using Illumina HiSeq 200.

研究目的：ADAR1介导的RNA编辑对造血发育至关重要。本研究的首要目标是，通过比对野生型小鼠的RNA测序数据与mm9参考基因组，鉴定A-to-I（G）错配位点，以此筛选ADAR1特异性RNA编辑位点，且该类位点在ADAR1E861A编辑缺陷型小鼠中未发生编辑或编辑频率降低；其次，明确ADAR1介导的A-to-I编辑缺失所带来的转录组学后果。
实验方法：本研究采用Illumina HiSeq2000测序平台，对胚胎第12.5天的野生型（WT）及ADAR1编辑缺陷型（ADAR1E861A）小鼠的胎肝mRNA表达谱进行测序，设置3次生物学重复。对通过质量过滤的序列读段，以转录本水平开展分析，先使用TopHat进行序列比对，再采用Cufflinks完成后续转录组分析。采用SYBR Green染料法进行qRT-PCR验证。参照Ramaswami G.等人2012年发表于《Nature Methods》的研究方法，使用Burrows-Wheeler比对工具（BWA）结合方差分析（ANOVA），从RNA-seq数据中鉴定A-to-I（G）RNA编辑位点。通过桑格测序对鉴定得到的RNA编辑位点进行验证。
实验结果：通过优化的数据分析流程，我们将每个样本约3000万条序列读段比对至小鼠基因组（构建版本mm9），并通过BWA在野生型与ADAR1E861A小鼠的胎肝中鉴定出14484个转录本。同一基因型的3次生物学重复间，RNA-seq数据的决定系数（R²）均大于0.94。在野生型与ADAR1E861A小鼠胎肝中，约4.4%的转录本呈现差异表达（LogFC≥1.5且p值<0.05）。与野生型小鼠相比，ADAR1E861A小鼠胎肝中的干扰素刺激基因出现显著上调，最高对数倍数变化可达11。在比对野生型与ADAR1E861A小鼠胎肝的RNA-seq数据后，共鉴定得到6012个A-to-I RNA编辑位点。
研究结论：本研究首次利用RNA测序技术，结合生物学重复，对胎肝转录组及A-to-I RNA编辑位点进行了系统性详细分析。A-to-I RNA编辑是ADAR1的核心功能，其可通过抑制内源RNA介导的干扰素信号通路发挥作用。本研究同样通过Illumina HiSeq 200测序平台，对胚胎第12.5天的野生型（WT）及ADAR E861A突变型小鼠的胎肝mRNA表达谱进行了3次重复的深度测序。